The C-C chemokine receptor 5 (CCR5) is a member of the G-protein-coupled, seven-transmembrane segment receptors (GPCRs), which comprise the largest superfamily of proteins in the body. In 1996, it was revealed that CCR5 represents one of the two essential co-receptors for HIV-1 entry into human CD4+ cells, thereby serving as an attractive target for possible intervention of infection by HIV-1 that uses CCR5 as a co-receptor (R5-HIV-1). The second extracellular loop (ECL2) of the GPCRs is known to play a critical role in ligand binding and ensuing signal transduction. The ECL2 of CCR5 is also thought to play an important role in CCR5 interactions with HIV-1 envelope. To date, scores of newly designed and synthesized CCR5 inhibitors have been reported to be potent against R5-HIV-1, and one such inhibitor, maraviroc, has recently been approved by the US Food and Drug Administration (FDA) for treatment of HIV-1-infected individuals who do not respond to any existing antiretroviral regimens. HIV-1-gp120 interacts with CCR5 following its binding to CD4, and such an interaction is thought to involve the V3 region of gp120 and the N-terminus and extracellular loops (ECLs) of CCR5. Recent reports have determined the orientation and location of CCR5 inhibitors within CCR5 and have shown that those inhibitors are all located in a hydrophobic cavity formed by the transmembrane domains of CCR5. In fact, earlier reports demonstrated that mutations in the extracellular loops did not have any effect on the binding of CCR5 inhibitors SCH-C and TAK-779. Taking these observations together, the binding sites in CCR5 for CCR5 inhibitors distinctly differ from the binding sites in CCR5 for HIV-1 gp120, strongly suggesting that CCR5 inhibitors block the interactions of CCR5 with HIV-1 gp120 through eliciting allosteric changes in ECL structures. We previously reported a small molecule CCR5 inhibitor, aplaviroc (APL), which has a high affinity to CCR5 (KD values of 3 nM) and exerts potent activity against a wide spectrum of R5-HIV-1 isolates, including multi-drug resistant R5-HIV-1 strains. APL significantly reduced viremia in patients with HIV-1 infection, as examined in a phase 2a clinical trial in the United States. However in phase 2b clinical trials with about 300 patients, four individuals receiving APL, developed Grade 3 or higher treatment-emergent elevations in ALT, and in late 2005, clinical development of APL was terminated. However, using APL as a specific probe, we further conducted structural analyses of CCR5 inhibitor interactions with CCR5, employing homology modeling, robust structure refinement, and molecular docking based on the site-directed mutagenesis-based saturation binding assay data of CCR5 inhibitors. In the current study, we determined the structural and molecular interactions of two novel CCR5 inhibitors, AK530 and AK317, both of which contain a novel spirodiketopiperazine (SDP) scaffold. We found that these two inhibitors lodge in a hydrophobic cavity located between the upper transmembrane domain and ECL2. We found substantial differences between the two molecules: AK530 had 10-fold greater CCR5-binding affinity (KD: 1.4 nM) than AK317 (16.7 nM), while their antiviral potency was virtually identical (IC50: AK530, 2.1 nM; AK317, 1.5 nM). Modeling analysis showed that AK530 has minimal interactions with S180 and K191 of ECL2, with which AK317 has a close association, suggesting that the interaction profile of the inhibitors with ECL2 residues is one of the important determinants of antiviral potency. We also found that the hairpin motif in the N-terminal half of ECL2 is critical for the HIV-1 envelope-elicited fusion event. The direct ECL2-engaging property of these inhibitors likely produces an ECL2 conformation, which HIV-1 gp120 cannot bind to, but also substantially delays HIV-1 from utilizing the "inhibitor-bound" CCR5 for cellular entry, a mechanism of HIV-1 resistance to CCR5 inhibitors. We carried out molecular dynamics simulations of inhibitor-unbound CCR5 and compared the conformation with inhibitor-bound CCR5. Critical inter-helix, and helix to extracellular loop hydrogen bond interactions seen in the unbound CCR5 were lost when transmembrane helix residues rearranged to accommodate AK530 and AK317 in the binding pocket. These observations add considerable insight to the mechanism of allosteric inhibition of CCR5-gp120 interaction by CCR5 inhibitors.